Protocol for detection of Salmonella Typhi and Salmonella Paratyphi A in Surface water (and other types of water samples)

The surface water does not require any processing and can be used directly with or without enrichment culture. The following steps describe bench preparation up to enrichment stage.

Put on gloves and spray hands with 70% ethanol. Rub hands together to sanitize all surfaces of the gloves.

Prepare your work surface by cleaning it with 70% ethanol.

Samples should arrive at the lab in a Whirl-Pak bag or glass bottles. Spray the outside of the bag/bottles with 70% ethanol and rub it well.

Rotate the Whirl-Pak bag or bottle with the sample five times to mix the sample.

The following steps are for optional enrichment of the sample. If enrichment is not performed, the sample can be used directly for membrane filtration and DNA extraction (Section 3).

In a biosafety hood, add 450mL of Universal Pre-enrichment (UP) Broth (USEPA Standard Analytical Protocol for Salmonella Typhi in Drinking Water) to a sterile 1 L flask.

Transfer 50mL of sample to the flask containing the UP Broth.

Incubate the flask in shaking incubator 37°C overnight (16-18 hr).

The following steps are for membrane filtration and DNA extraction. They can be performed following step 1.4 or step 2.3.

Clean your workspace and set up your filter units.

Transfer the folded filter to a bead tube (from Qiagen DNeasy PowerWater kit).

Add 1mL of Buffer PW1 (Qiagen DNeasy PowerWater kit) to the bead tube and vortex for 0h 5m 0s.

Proceed with the DNA Extraction according to manufacturer’s protocol (Qiagen DNeasy PowerWater kit).

Using sterile forceps, place a clean membrane filter on the base of the filter unit.

Place the cup on top of the filter.

Add 20mL of enriched sample to the cup and turn on the vacuum.

Allow the sample to filter until liquid is no longer visible on the filter.

Turn off the vacuum and remove the cup from the base.

Using a sterile forcep, remove the filter from the base.

Using two sterile forceps, fold the filter in half inward, so the cells are now contained inside the folded filter.

Fold the filter in half again.

Test DNA extracts for S . Typhi and S . Paratyphi A using Taqman-based quantitative real-time PCR (qPCR) platform.

Detection of S . Typhi

S . Typhi is detected using duplex PCR protocol developed by researchers at the University of Washington (Scott Meschke and team) usIng primers and probes targeting the tviB and staG genes (Nair et al., 2019).

tviB _F 5’TGTGGTAAAGGAACTCGGTAAA-3’;

tvi B_R 5’-GACTTCCGATACCGGGATAATG-3’;

tvi B_P HEX - TGGATGCCGAAGAGGTAAGACGAGA-BHQ1;

sta G ___ F 5’- CGCGAAGTCAGAGTCGACATAG-3’;

sta G ___ R 5’-AAGACCTCAACGCCGATCAC-3’;

sta G ___ P FAM-5’-CATTTGTTCTGGAGCAGGCTGACGG-3’-BHQ1

The reaction mixture contain 0.65 µl of tviB_F (20µM), 0.75 µl each of tviB_R (20µM), staG_F(20µM), and staG_R (20µm), 0.5 µl each of the probe tviB_P (10µM) and staG_P (10 µM), 12.5 µl of SsoAdvanced Universal Probes Supermix (Bio-rad), and 5 µl of DNA in a final volume of 25 µl. The PCR reaction conditions include initial denaturation at 95^oC for 5 min, followed by 45 cycles of 95^oC 30 sec, 64^oC 30 sec, 72^oC 10 sec, and final extension at 72^oC for 5 min.

Detection of S . Paratyphi A

S . Paratyphi A Is detected using primers and probe targeting SPA2308 (Nga et al., 2010)

SPA2308_F 5’- ACGATGATGACTGATTTATCGAAC-3’;

SPA2308_R5’-TGAAAAGATATCTCTCAGAGCTGG-3’;

SPA2308_PCY5 - CCCATACAATTTCATTCTTATTGAGAATGCGC-BHQ2

The reaction mixture containing 1 µl of each primer (10µM), 0.4 µl of probe (10µM), 200µM of dNTPs, 5mM of MgCl2, 5U of HotStar Taq DNA polymerase (Qiagen), and 5 µl of DNA in a final reaction volume of 25 µl. The PCR reaction conditions include initial denaturation at 95^oC for 5 min, followed by 45 cycles of 95^oC 30 sec, 60^oC 30 sec, 72^oC 30 sec, and final extension at 72^oC for 10 min.